Animal models of Alzheimer's disease (AD) recapitulate early phases of the human condition, but not the later stages of neuronal death and dementia. These models develop histologically similar, but distinguishable A beta amyloid pathology. The imaging ligand Pittsburgh Compound B (PIB) binds insoluble A beta from human, animal and synthetic systems with nM affinity. However, the stoichiometry of PIB binding to human AD brain is approximately 500- to 10,000-fold greater than that in animal models or synthetic A beta fibrils. Other A beta ligands such as Thioflavin S and Congo Red do not discriminate between human, animal, and synthetic A beta fibrils. We hypothesize that the difference in PIB binding between human brain and animal A beta fibrils is integral to the mechanism of neuronal death and dementia in human AD, and contributes to the lack of complete recapitulation of the disease in animal models. Preliminary data indicate that uncharacterized biochemical components in addition to A beta are required for the high binding stoichiometry of PIB in AD brain, which resides in a denaturation-resistant assembly. The proposed work will generate photoaffinity probes for the human A beta PIB binding site that also contain a tag to enable affinity purification and we will utilize these probes to affinity isolate nd identify protein and lipid components contacting the PIB ligand in the AD brain by mass spectral analysis. Specific Aim 1. Synthesize and optimize a series of photoaffinity ligands with handles for affinity tags that are selective for the PIB binding site of human AD brain. Specific Aim 2. Identify the protein and lipid components that form the PIB ligand binding site of AD brain using MR-PIB and mass spectrometry. The long term goal of this project is to use the identified components and structure of the human AD A beta fibril and its PIB ligand binding pocket to provide insight into unique properties of human AD A beta assemblies that contribute to AD. Ultimately we will determine the differences between human and AD animal models A beta assemblies to elucidate AD-related cellular and environmental mechanisms that will inform the improvement of animal models and identify potential novel drug targets.